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Modulation of u-PA, MMPs and their inhibitors by a novel nutrient mixture in pediatric human sarcoma cell lines

M.Waheed Roomi, Tatiana Kalinovsky, Aleksandra Niedzwiecki* and Matthias Rath
Dr. Rath Research Institute, Santa Clara, CA
International Journal of Oncology 2013; 43: 1027-1035

Pediatric sarcomas are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the ECM and basement membrane, allowing cancer cells to spread to distal organs. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs).

Our main objective was to study the effect of a nutrient mixture (NM) on activity of u-PA, MMPs and TIMPs in various human pediatric sarcomas.  Human osteosarcoma MNNG-HOS, osteosarcoma U-2OS and rhabdomyosarcoma RD cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000 µg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. All sarcoma cell lines studied expressed u-PA, which was inhibited by NM in a dose-dependent manner. On gelatinase zymography, osteosarcoma MNNG-HOS showed a band corresponding to MMP-2 and induction of MMP-9 with PMA (100 ng/ml) treatment. Osteosarcoma U-2OS showed strong bands corresponding to inactive MMP-2 and MMP-9 and faint bands corresponding to active MMP-2 and MMP-9 dimer; PMA treatment enhanced MMP-9 and MMP-9 dimer activity. Rhabdomyosarcoma showed MMP-2 and faint MMP-9 bands; PMA treatment enhanced MMP-9 expression. NM inhibited their expression in a dose-dependent manner.  Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose–dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA /MMPs and TIMPs. These findings suggest the therapeutic potential of NM in treatment of pediatric sarcomas.

Running Title: 
Modulation of pediatric sarcoma cancer cell line MMPs, u-PA, and TIMPs activity

Key words: 
osteosarcoma MNNG-HOS and U-2OS, rhabdomyosarcoma RD, u-PA, MMP-2 and MMP-9, TIMP-2, PMA, nutrient mixture